Glycerol 3 phosphate

[¹³C]CDP-Gro was synthesized as described previously^{43 (link)} with slight modifications. Briefly, His-tagged AQ1368, a glycerol-phosphate cytidylyltransferase from Aquifex aeolicus, was expressed in E. coli BL21(DE3), purified using nickel-chelate chromatography (HisTrap HP 5 mL, GE Healthcare), and dialyzed with 50 mM Tris-HCl (pH 8.6) containing 150 mM NaCl and 5 mM MgCl₂. [¹³C]CDP-Gro was produced by heating 20 μL of solution containing 50 mM Tris-HCl (pH 8.6), 5 mM MgCl₂, 6.25 μg His-AQ1368, 2.5 mM CTP (Cytidine-13C9; Merck Sigma), and 2.5 mM glycerol-3-phosphate (Merck Sigma) at 37 °C for 5 min. The product was separated using reverse-phase HPLC with a COSMOSIL 5C18-AR-II column (4.6 × 250 mm; Nacalai Tesque) and isocratic elution with 20 mM TEAA buffer (pH 7). Product elution was monitored by measuring the absorbance at 260 nm. [¹³C]CDP-Gro was then collected, dried under vacuum, and dissolved in water. [¹³C]CDP-Gro production was confirmed by LC-MS analysis, as described above.

S1QEL1.1, S1QEL2.1 [5 (link)], S1QEL1.719 [25 ] and S3QEL3 [6 (link)] were provided by Calico Life Sciences LLC (South San Francisco, CA) and AbbVie Inc. (Chicago, IL). They were maintained as 10 mM stocks in dimethylsulfoxide at room temperature, shielded from bright light and diluted in dimethylsulfoxide as required before use. Amplex UltraRed (Cat No. A36006) was from ThermoFisher; atpenin A5 (Cat No. 11898) and FCCP (Cat No. 15218) from Cayman Chemicals; piericidin A (Cat No. 2738-64-9) from Santa Cruz Biotechnology; and horseradish peroxidase (HRP) (Cat No. P8125), superoxide dismutase 1 (SOD1) (Cat No. S7571), rotenone (Cat No. R8875), myxothiazol (Cat No. T5580), succinate (Cat No. 14160), glycerol 3-phosphate (Cat No. 94124), malonate (Cat No. 792535) and glutamate (Cat No. 1294976) were from Sigma.

The oxygen consumption rate was determined using the Oroboros Oxygraph-2K (Oroboros Instruments Corp). For each sample, 2 × 10⁷ cells were harvested and washed once with Mir05 mitochondrial respiration medium (0.5-mM EGTA, 3-mM MgCl₂, 60-mM lactobionic acid, 20-mM taurine, 10-mM KH₂PO₄, 20-mM Hepes, 110-mM sucrose, 1 mg/ml fatty acid–free bovine serum albumin, pH 7.1). The cell pellet was resuspended in 2.1 ml of Mir05 and transferred into the respiration chamber at the appropriate growth temperature for each life stage and under constant stirring. In the experiments carried out with intact PCF cells, 10-mM glycerol-3-phosphate (Sigma, 17766) was added, and complex IV- and AOX-mediated respirations were inhibited by injection of 1-mM KCN and 250-μM SHAM (Sigma S607), respectively. For the experiments performed with permeabilized BSF cells, the addition of 4-μM digitonin (Sigma D141) preceded the injection of 20-mM glycerol-3-phopshate into the chamber, and respiratory inhibition was achieved by addition of 250-μM SHAM. The most stable portion of either the oxygen consumption rate slope (PCF experiments) or the oxygen concentration in the chamber slope (BSF experiments) was determined for each biological replicate after the addition of substrates and inhibitors. The values were plotted and analyzed statistically using GraphPad Prism 8.0 software.

Activity staining of mGPDH in native gels was performed according to a modified protocol originally described in [27 (link)]. Gel slices were stained using solution of 5 mM Tris–HCl (pH 7.4), 3 mM MgCl₂, 0.88 mM menadione, 1.2 mM NitroBlue Tetrazolium, 1.5 μM rotenone, 2 mM KCN and 10 mM glycerol-3-phosphate (Merck/Sigma, 61668) for 1 h. Subsequently gels were denatured in 50% methanol/10% acetic acid for 15 min, fixed in 10% acetic acid for 10 min and scanned on a flatbed scanner.

For differentiation to osteocyte, passage 3 BM-MSCs and passage 3 AT-MSCs were incubated to differentiate into osteoblasts in corresponding induction medium for 2 weeks. The cells were maintained in control and osteogenic media. The control medium consisted of DMEM (Gibco, USA), supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). The osteogenic medium contained DMEM, 15% FBS (Gibco, USA), 100 μM l-ascorbic acid, 10 mM glycerol 3-phosphate, and 100 nM dexamethasone (Sigma-Aldrich). The medium was replaced 2 times a week. After 2 weeks the cultured cells were stained with Alizarin Red Solution to visualize calcium deposits. The cells were fixed in 4% paraformaldehyde (PFA) for 20 min and stained using Alizarin Red Solution for 20 min at room temperature.